Stem Cell, Precursor Cell, or Target Cell-Based Treatment of Multi-Organ Failure and Renal Dysfunction

ABSTRACT

Methods for the treatment of acute renal failure, multi-organ failure, early dysfunction of kidney transplant, chronic renal failure, organ dysfunction, and wound healing are provided. The methods include delivering a therapeutic amount of hematopoietic stem cells, non-hematopoietic, mesenchymal stem cells, hemangioblasts, and pre-differentiated cells to a patient in need thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/551,317, filed Sep. 29, 2005, which a National Stage of InternationalApplication No. PCT/US2004/009922, filed Mar. 31, 2004, which claimspriority to U.S. Provisional Application No. 60/459,554, filed Apr. 1,2003 and U.S. Provisional Application No. 60/475,178, filed Jun. 2,2003, which are hereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The subject matter described herein was in-part made possible by supportfrom the Department of Veterans Affairs. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to methods for treating organdysfunction, multi-organ failure and renal dysfunction, including, butnot limited to acute renal failure, transplant associated acute renalfailure, chronic renal failure, and wound healing. More specifically,the inventive methods use stem cells, precursor cells or organ-specifictarget cells and combinations thereof

BACKGROUND OF THE INVENTION

Multi-organ failure (MOF) remains a major unresolved medical problem.MOF develops in the most severely ill patients who have sepsis,particularly when the latter develops after major surgery or trauma. Itoccurs also with greater frequency and severity in elderly patients,those with diabetes mellitus, underlying cardiovascular disease andimpaired immune defenses. MOF is characterized by shock, acute renalfailure (ARF), leaky cell membranes, dysfunction of lungs, liver, heart,blood vessels and other organs. Mortality due to MOF approaches 100%despite the utilization of the most aggressive forms of therapy,including intubation and ventilatory support, administration ofvasopressors and antibiotics, steroids, hemodialysis and parenteralnutrition. Many of these patients have serious impairment of the healingof surgical or trauma wound, and, when infected, these wounds furthercontribute to recurrent infections, morbidity and death.

ARF is defined as an acute deterioration in renal excretory functionwithin hours or days, resulting in the accumulation of “uremic toxins,”and, importantly, a rise in the blood levels of potassium, hydrogen andother ions, all of which contribute to life threatening multisystemcomplications such as bleeding, seizures, cardiac arrhythmias or arrest,and possible volume overload with pulmonary congestion and poor oxygenuptake. The most common cause of ARF is an ischemic insult of the kidneyresulting in injury of renal tubular and postglomerular vascularendothelial cells. The principal etiologies for this ischemic form ofARF include intravascular volume contraction, resulting from bleeding,thrombotic events, shock, sepsis, major cardiovascular surgery, arterialstenoses, and others. Nephrotoxic forms of ARF can be caused byradiocontrast agents, significant numbers of frequently used medicationssuch as chemotherapeutic drugs, antibiotics and certainimmunosuppressants such as cyclosporine. Patients most at risk for allforms of ARF include diabetics, those with underlying kidney, liver,cardiovascular disease, the elderly, recipients of a bone marrowtransplant, and those with cancer or other debilitating disorders.

Both ischemic and nephrotoxic forms of ARF result in dysfunction anddeath of renal tubular and microvascular endothelial cells. Sublethallyinjured tubular cells dedifferentiate, lose their polarity and expressvimentin, a mesenchymal cell marker, and Pax-2, a transcription factorthat is normally only expressed in the process of mesenchymal-epithelialtransdifferentiation in the embryonic kidney. Injured endothelial cellsalso exhibit characteristic changes.

The kidney, even after severe acute insults, has the remarkable capacityof self-regeneration and consequent re-establishment of nearly normalfunction. It is thought that the regeneration of injured nephronsegments is the result of migration, proliferation and redifferentationof surviving tubular and endothelial cells. However, theself-regeneration capacity of the surviving tubular and vascularendothelial cells may be exceeded in severe ARF. Patients with isolatedARF from any cause, i.e., ARE that occurs without MOF, continue to havea mortality in excess of 50%. This dismal prognosis has not improveddespite intensive care support, hemodialysis, and the recent use ofatrial natriuretic peptide, Insulin-like Growth Factor-I (IGF-I), morebiocompatible dialysis membranes, continuous hemodialysis, and otherinterventions. An urgent need exists to enhance the kidney'sself-defense and autoregenerative capacity after severe injury.

Another acute form of renal failure, transplant-associated acute renalfailure (TA-ARF), also termed early graft dysfunction (EGD), commonlydevelops upon kidney transplantation, mainly in patients receivingtransplants from cadaveric donors, although TA-ARF may also occur inpatients receiving a living related donor kidney. Up to 50% of currentlyperformed kidney transplants utilize cadaveric donors. Kidney recipientswho develop significant TA-ARF require treatment with hemodialysis untilgraft function recovers. The risk of TA-ARE is increased with elderlydonors and recipients, marginal graft quality, significant comorbiditiesand prior transplants in the recipient, and an extended period of timebetween harvest of the donor kidney from a cadaveric donor and itsimplantation into the recipient, known as “cold ischemia time.” Earlygraft dysfunction or TA-ARF has serious long-term consequences,including accelerated graft loss due to progressive, irreversible lossin kidney function that is initiated by TA-ARF, and an increasedincidence of acute rejection episodes leading to premature loss of thekidney graft. Therefore, a great need exists to provide a treatment forearly graft dysfunction due to TA-ARF or EGD.

Chronic renal failure (CRF) or Chronic Kidney Disease (CKD) is theprogressive loss of nephrons and consequent loss of renal function,resulting in End Stage Renal Disease (ESRD), at which time patientsurvival depends on dialysis support or kidney transplantation. Theprogressive loss of nephrons, i.e., glomeruli, tubuli andmicrovasculature, appears to result from self-perpetuating fibrotic,inflammatory and sclerosing processes, most prominently manifested inthe glomeruli and renal interstitium. The loss of nephrons is mostcommonly initiated by diabetic nephropathy, glomerulonephritides, manyproteinuric disorders, hypertension, vasculitic, inflammatory and otherinjuries to the kidney. Currently available forms of therapy, such asthe administration of angiotensin converting enzyme inhibitors,angiotensin receptor blockers, other anti-hypertensive andanti-inflammatory drugs such as steroids, cyclosporine and others, lipidlowering agents, omega-3 fatty acids, a low protein diet, and optimalweight, blood pressure and blood sugar control, particularly indiabetics, can significantly slow and occasionally arrest the chronicloss of kidney function in the above conditions. The development of ESRDcan be prevented in some compliant patients and delayed others. Despitethese successes, the annual growth of patient numbers with ESRD,requiring chronic dialysis or transplantation, remains at 6%,representing a continuously growing medical and financial burden. Thereexists an urgent need for the development of new interventions for theeffective treatment of CRF or CKD and thereby ESRD, to treat patientswho fail to respond to conventional therapy, i.e., whose renal functioncontinues to deteriorate. Stem cell treatment will be provided toarrest/reverse the fibrotic processes in the kidney.

Taken together, therapies that are currently utilized in the treatmentof ARF, the treatment of established ARF of native kidneys per se or aspart of MOF, and ARF of the transplanted kidney, and organ failure ingeneral have not succeeded to significantly improve morbidity andmortality in this large group of patients. Consequently, there exists anurgent need for the improved treatment of MOF, renal dysfunction, andorgan failure.

Very promising pre-clinical studies in animals and a few early phaseclinical trials administer bone marrow-derived hematopoietic stem cellsfor the repair or protection of one specific organ such as the heart,small blood vessels, brain, spinal cord, liver and others. Thesetreatments have generally used only a single population of bone-marrowstem cells, either Hematopoietic (HSC) or Mesenchymal Stem Cells (MSC),and obtained results are very encouraging in experimental stroke, spinalcord injury, and myocardial infarction. The intracoronary administrationof stem cells in humans with myocardial infarction or coronary arterydisease has most recently been reported to result in significant adverseevents such as acute myocardial infarction, other complications anddeath. Peripheral administration of stem cells or the direct injectioninto the injured myocardium showed more favorable results both in animaland Phase I trials. MSC have been infused into patients a few weeksafter they first received a bone marrow transplant in the treatment ofcancers, leukemias, osteogenesis imperfecta, and Hurler's syndrome toaccelerate reconstitution of adequate hematopoiesis. Effective treatmentof osteogenesis imperfecta and Hurler's syndrome has been shown usingMSC. Importantly, administration of a mixture of HSC and MSC, known tophysiologically cooperate in the maintenance of hematopoiesis in thebone marrow, has, until now (see below) not been utilized for thetreatment of any of the above listed renal disorders, MOF or woundhealing.

In ARF (native kidneys, transplanted kidney), microvascular endothelialcells and proximal as well as distal tubular cells become dysfunctionaland are destroyed when injured, insults that together mediate the acuteloss of kidney function. Successful recovery from ARF depends directlyon the repair of injured renal microvessels and tubular segments. Sinceboth HSC and MSC possess a remarkable level of plasticity, i.e., arecapable to differentiate into several non-hematopoietic cell types(neurons, heart, muscle, liver, vascular and other cells) includingrenal tubular and vascular endothelial cells, pre-clinical studies werebegun to test the concept that the co-administration of HSC and MSC maybe more renoprotective than the administration of either HSC or MSCalone, as it reproduces their mutually supportive capacity in the bonemarrow. Studies demonstrated that MSC can be induced, using co-culture,conditioned media and injury models, to differentiate in vitro both intovascular endothelial and tubular cell phenotypes. In addition, syngeneicvascular endothelial cells (EC) or EC derived from MSC were tested todetermine whether EC could function in rats with ARF as kidneyprotective renal EC precursors. Without wishing to be bound to anyparticular theory, the present inventor believes that microvasculardysfunction and EC injury and death are prominent mediators ofinadequate renal blood flow in ARF, and that the delivery of “healthy”EC or their precursors could improve renal hemodynamics, therebyaugmenting tubular cell survival, protecting renal function andhastening tissue repair. The results of these studies to date show: (1)all types of EC or EC precursors, derived from all tested sources,significantly protect renal function and improve outcome in rats withestablished ischemic ARE, reducing mortality from ˜40% to <5%; (2) MSCadministration alone results in delayed but significantly acceleratedrecovery of renal function; (3) HSC infusion alone shows similar orslightly less improvement in functional recovery compared to thatobtained with MSC; (4) the administration of a defined mix of HSC andMSC, as discussed below, appears highly effective in the treatment ofARF, the rapid reestablishment of adequate renal function after ARF, andessential elimination of animal mortality.

In the kidney, the administration of pluripotent stem cells, derivedfrom hematopoietic or non-hematopoietic sources, can be utilized forrepair of critically damaged kidney tissues. The physical or functionalloss of reno-vascular endothelial and tubular cells and thus renalfunction, whether occurring in acute or chronic renal failure, is aserious medical condition that will be ameliorated by the presentinvention. Any slowing, arrest, or reversal of the decline in renalfunction provided by the present invention will be enormously beneficialto the affected patients with ARF, TA-ARF, CRF, or any kidneyfailure-associated systemic dysfunction, MOF or wound healing.

BRIEF SUMMARY

In order to alleviate one or more shortcomings of the prior art, methodsof treatment are provided herein. In accordance with the presentinvention, methods for the treatment of acute renal failure, multi-organfailure, and early dysfunction of kidney transplant, chronic renalfailure, organ dysfunction, or wound healing are provided.

In one aspect of the present invention, a method of treatment for acuterenal failure, multi-organ failure, early dysfunction of kidneytransplant, chronic renal failure, organ dysfunction, or wound healingis provided. The method includes delivering a therapeutic amount of amixture of hematopoietic stem cells and/or mesenchymal stem cells to apatient in need thereof.

In another aspect of the present invention, a method of treating acuterenal failure, multi-organ failure, early dysfunction of kidneytransplant, chronic renal failure, or wound healing is provided. Themethod includes delivering a therapeutic amount of pre-differentiatedstem cells to a patient in need thereof. The cells arepre-differentiated in vitro into kidney- or other organ-specific cells.

In another aspect of the present invention, a method of treating acuterenal failure, multi-organ failure, early dysfunction of kidneytransplant, chronic renal failure, or wound healing is provided. Themethod includes delivering a therapeutic amount of hemangioblasts to apatient in need thereof.

In yet another aspect of the present invention, a method of treatingkidney dysfunction is provided. The method includes delivering atherapeutic amount of non-transformed stem cells to a patient in needthereof.

In yet another aspect of the present invention, a method of treatingkidney dysfunction is provided. The method includes delivering atherapeutic amount of genetically modified stem cells to a patient inneed thereof.

In another aspect of the present invention, a composition is provided.The composition includes a therapeutic amount of hematopoietic stemcells and mesenchymal stem cells.

In yet another aspect of the present invention, a method of treatingkidney dysfunction is provided. The method includes delivering atherapeutic amount of a stimulant of stem cell mobilization to a patientin need thereof. The stimulant mobilizes stem cells to the kidney.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will utilize stem cells for the repair of damagedtissues in patients in need thereof and prevention of further injury.The term “stem cells” as used herein refers to cells havingdevelopmental plasticity that are able to produce other cell types thanthe cells from which the stem cells are derived. The terms “stem cells”and “pluripotent stem cells” refer to cells that are not fixed as topotential development. The stem cells of the present invention arenon-embryonic in origin, preferably adult stem cells.

In one aspect of the present invention, autologous hematopoietic stemcells (HSC) or mesenchymal stem cells (MSC) are administered or, whenneeded, are co-administered to a patient in need thereof in definedratios. The administration of HSC or MSC or co-administration of HSC andMSC may be used in the treatment of multi-organ failure, acute renalfailure of native kidneys, ARF of native kidneys in multi-organ failure,ARF in transplanted kidneys, kidney dysfunction, organ dysfunction andwound repair. HSC and MSC may be used to treat additional disordersknown to one of skill in the art. Defined ratios of HSC and MSC may beused to treat the dysfunction of other organs including transplantedorgans, such as, but not limited to, lungs, liver, heart, or poorlyhealing wounds. Allogeneic MSC or HSC may also be administered alone orco-administered in defined ratios to be utilized as treatment, forexample in multi-organ failure, all types of renal dysfunction, organdysfunction, and to promote wound healing. The term “treatment” as usedherein refers to stem cells delivered to repair an injured organ and toprevent further injury in a patient in need thereof.

The conditions identified for stem cell treatment in patients in needthereof, including, but not limited to, multi-organ failure, acute renalfailure of native kidneys, ARF of native kidneys in multi-organ failure,ARF in transplanted kidneys, kidney dysfunction, organ dysfunction andwound repair refer to conditions known to one of skill in the art.Descriptions of these conditions may be found in medical texts, such asThe Kidney, by Barry M. Brenner and Floyd C. Rector, Jr., W B SaundersCo., Philadelphia, last edition, 2001, which is incorporated herein inits entirety by reference.

ARF is defined as an acute deterioration in renal excretory functionwithin hours or days. In severe ARF, the urine output is absent or verylow. As a consequence of this abrupt loss in function, azotemiadevelops, defined as a rise of serum creatinine levels and blood ureanitrogen levels. Serum creatinine and blood urea nitrogen levels aremeasured. When these levels have increased to approximately 10 foldtheir normal concentration, this corresponds with the development ofuremic manifestations due to the parallel accumulation of uremic toxinsin the blood. The accumulation of uremic toxins causes bleeding from theintestines, neurological manifestations most seriously affecting thebrain, leading, unless treated, to coma, seizures and death. A normalserum creatinine level is ˜1.0 mg/dL, a normal blood urea nitrogen levelis ˜20 mg/dL. In addition, acid (hydrogen ions) and potassium levelsrise rapidly and dangerously, resulting in cardiac arrhythmias andpossible cardiac standstill and death. If fluid intake continues in theabsence of urine output, the patient becomes fluid overloaded, resultingin a congested circulation, pulmonary edema and low blood oxygenation,thereby also threatening the patient's life. One of skill in the artinterprets these physical and laboratory abnormalities, and bases theneeded therapy on these findings.

MOF is a condition in which kidneys, lungs, liver and heart functionsare generally impaired simultaneously or successively, resulting inmortality rates as high as 100% despite the conventional therapiesutilized to treat ARF. These patients frequently require intubation andrespirator support because their lungs develop Adult RespiratoryDistress Syndrome (ARDS), resulting in inadequate oxygen uptake and CO2elimination. MOF patients also depend on hemodynamic support,vasopressor drugs, and occasionally, an intra-aortic balloon pump, tomaintain adequate blood pressures since these patients are usually inshock and suffer from heart failure. There is no specific therapy forliver failure which results in bleeding and accumulation of toxins thatimpair mental functions. Patients may need blood transfusions andclotting factors to prevent or stop bleeding. MOF patients will be givenstem cell therapy when the physician determines that therapy is neededbased on assessment of the patient.

EGD or TA-ARF is ARF that affects the transplanted kidney in the firstfew days after implantation. The more severe TA-ARF, the more likely itis that patients will suffer from the same complications as those whohave ARF in their native kidneys, as above. The severity of TA-ARF isalso a determinant of enhanced graft loss due to rejection(s) in thesubsequent years. These are two strong indications for the prompttreatment of TA-ARF with the stem cells of the present invention.

Chronic renal failure (CRF) or Chronic Kidney Disease (CID) is theprogressive loss of nephrons and consequent loss of renal function,resulting in End Stage Renal Disease (ESRD), at which time patientsurvival depends on dialysis support or kidney transplantation. Need forstem cell therapy of the present invention will be determined on thebasis of physical and laboratory abnormalities described above.

Stem cell therapy will preferably be given to patients in need thereofwhen one of skill in the art determines that conventional therapy fails.Conventional therapy includes hemodialysis, antibiotics, blood pressuremedication, blood transfusions, intravenous nutrition and in some cases,ventilation on a respirator in the ICU. Hemodialysis is used to removeuremic toxins, improve azotemia, correct high acid and potassium levels,and eliminate excess fluid. Stem cell therapy of the present inventionis not limited to treatment once conventional therapy fails and may begiven immediately upon developing an injury or together withconventional therapy.

Monitoring patients for a therapeutic effect of the stem cells deliveredto a patient in need thereof and assessing further treatment will beaccomplished by techniques known to one of skill in the art. Forexample, renal function will be monitored by determination of bloodcreatinine and BUN levels, serum electrolytes, measurement of renalblood flow (ultrasonic method), creatinine and inulin clearances andurine output. A positive response to therapy for ARF includes return ofexcretory kidney function, normalization of urine output, bloodchemistries and electrolytes, repair of the organ and survival. For MOF,positive responses also include improvement in blood pressure andimprovement in functions of one or all organs.

In another aspect of the present invention, bone-marrow derived or stemcells derived from other organs may be used to treat critically damagedkidney tissues and to prevent damage to kidney tissue in patients atrisk for developing kidney damage. A single stem cell population orcombinations of stem cell populations or stem cells that arepre-differentiated into kidney-specific precursor cells (e.g., tubular,vascular endothelial and glomerular cells, etc.) may be used to treat orprevent kidney damage.

Stem cells may be utilized to effectively repopulate dead ordysfunctional kidney cells because of the “plasticity” of stem cellpopulations. The term “plasticity” refers to the phenotypically broaddifferentiation potential of cells that originate from a defined stemcell population. Stem cell plasticity can include differentiation ofstem cells derived from one organ into cell types of another organ.“Transdifferentiation” refers to the ability of a fully differentiatedcell, derived from one germinal cell layer, to differentiate into a celltype that is derived from another germinal cell layer.

It was assumed, until recently, that stem cells gradually lose theirpluripotency and thus their differentiation potential duringorganogensis. It was thought that the differentiation potential ofsomatic cells was restricted to cell types of the organ from whichrespective stem cells originate. This differentiation process wasthought to be unidirectional and irreversible. However, recent studieshave shown that somatic stem cells maintain some of theirdifferentiation potential. For example, hematopoietic stem cells areable to transdifferentiate into muscle, neurons, liver, myocardialcells, and kidney. It is possible that as yet undefined signals thatoriginate from injured and not from intact tissue act astransdifferentiation signals.

The present invention will utilize pluripotent stem cell populations totreat renal dysfunction and other organ dysfunction. Stem cells,including HSC, MSC, cells derived from MSC by pre-differentiation(organ-specific progenitor cells of target organs) will be used, aloneor in combinations thereof, in order to augment the kidney'sautoprotective capacity and to support and boost the repair processes inpatients with renal dysfunction and other organ dysfunction. Stem cellsused in the present intervention express receptors that, when activatedby chemokine signals that emanate from sites of injury in the damagedorgans, result in the horning of stem cells to these injury sites. Theadministration of a single cell type or mixes thereof results in thelocalized delivery to and accumulation of stem cells at the sites ofinjury. Since stem cells express renotropic survival factors,anti-inflammatory cytokines, vasoactive and other beneficial factors,these are released in the microenvironment of the injury sites in thekidney or other organs. The local levels of protective humoral factorsare optimized and immediate beneficial actions on renal and other organfunction are elicited. In subsequent steps, delivered stem cells andother cells gradually integrate as progressively differentiated targetcells into the injured tubular epithelium and/or endothelium, anddirectly participate in the cellular repair processes. The pluripotentstem cell populations used to protect and repair the dysfunctionalkidney and other organs may be derived from hematopoietic or mesenchymalstem cells, as hemangioblasts, as EC progenitors, or from other organssuch as kidney, liver, muscle, or fat. Other cells and organs such asumbilical cord blood or cells may provide a source of stem cells toprotect and repair dysfunctional kidneys and other organs. The term“non-transformed” as used herein refers to stem cells that have not beengenetically modified with exogenous DNA or RNA.

In one embodiment of the present invention, the pluripotent stem cellpopulation is derived from HSC. The HSC are derived from the bone marrowor peripheral blood, preferably the bone marrow. The HSC are isolatedfrom a healthy and compatible donor or the patients themselves bytechniques commonly known in the art. The HSC population may be enrichedfor pluripotent HSC using fluorescence activated cell sorting (FACS) orother methods. The pluripotent HSC may be enriched by FAGS by selectingfor “c-kit” positive, “sca-1” positive and “lin negative” cells. “c-kit”and “sca-1” cells are known to one of skill in the art as beingreceptors known to be on the surface of stem cells. A “lin negative”cell is known to one of skill in the art as being a cell that does notexpress antigens characteristic of specific cell lineages and thus ismore primordial, pluripotent and capable of self-renewal. The HSC may beCD 34 positive or negative. Any method known to one of skill in the artmay be used to enrich the population of pluripotent stem cells from thewhole population of bone marrow cells, and, if necessary, cryopreservethem until needed for therapy.

Alternatively and time permitting, autologous HSC may be obtained fromthe peripheral blood using routine HSC mobilization protocols known toone of skill in the art with repeated leukapheresis. HSC may be enrichedby FACS, and preserved until use. Mobilization of HSC into theperipheral circulation is accomplished by the daily administration ofG-CSF alone or in conjunction with cytoxan or SCF. Doses of G-CSF,cytoxan and SCF for mobilization of HSC known to one of skill in theart. Mobilization doses, by way of example, may be the same doses usedin the treatment of autologous bone marrow transplant patients. Theresultant increase in peripheral leukocytes is paralleled by an increasein circulating HSC numbers which are collected by repeatedleukapheresis. This “slower” approach of collecting HSC may be bestsuited for those patients who are scheduled to undergo an elective highrisk surgery, i.e., patients in whom there is sufficient time to collectHSC in this fashion, and if used in combination with MSC, while theirMSC are conventionally obtained from their bone marrow aspirate (seebelow).

MSC for administration preferably are derived from bone marrow aspiratesthat are placed into sterile culture in vitro. MSC from the bone marrowaspirates adhere to the bottom of a culture dish while essentially allother cell types remain in suspension. (Friedenstein, Exp. Heinatol.4:267-74, 1976). After discarding the non-adherent cells, MSC will growand expand in culture, yielding a well defined population of pluripotentstem cells. After expansion in vitro, collected MSC may be furtherdepleted of CD 45 positive cells, by FACS, to remove residualmacrophages or other hematopoietic cell lineages prior theiradministration to the patient. MSC may be derived from the patient, froma compatible donor, or from a blood group compatible but allogeneicdonor, exploiting in the latter case the immunomodulating capacity ofMSC (see below). Donor stem cells may be used from a donor havingsimilar compatibility as defined for the organ transplantation, known toone skilled in the art. Since MSC can be expanded in vitro, thetreatment regimen with MSC can be easily repeated in order to furtheraugment the cellular repair processes in the injured kidney. Any methodknown to one of skill in the art may be used to enrich the population ofpluripotent MSC from the whole population of bone marrow cells, and, ifnecessary, cryopreserve them until needed for therapy.

Any donor can be used as a source of stem cells. Preferably, autologousstem cells are used since they eliminate concerns regarding immunetolerance. Additionally, by way of example, repetitive administrationsof autologous MSC and HSC are possible.

MSC may also be used (see above). MSC have been shown to suppress theT-cell response, remaining immunomodulating even after differentiationinto various cell types. MSC do not elicit an immune response that wouldresult in their rejection by the donor. Suppression of the MSC responsemakes MSCs suitable as a first line intervention in patients in needthereof, requiring only assurance of blood group compatibility betweenMSC donor and recipient. Reasons for administering allogeneic MSCinclude:

(a) Suitability, despite being allogeneic cells, for immediateadministration to a blood group compatible patient in need thereof. Thisis based on the inherent immunomodulating capacity that MSC andMSC-derived cells possess. MSC may be collected and saved for “off theshelf” use to provide an immediate source of cells for infusion whenneeded. Autologous MSC, in contrast, require more time for collection,enrichment and expansion of the cells and are not immediately available.Immediate availability of MSC is significant in patients with the mostsevere forms of ARF and MOF.

(b) Bone marrow in a patient in need of stem cell therapy may be a poorsource of adequate numbers of stem cells. The patient may have receivedbone marrow toxic drugs or radiation or may have bone marrow cancer,thereby making the patient's own MSC unusable.

(c) A patient may refuse or may not be able to consent to the harvestingof his/her own bone marrow cells.

(d) Bone marrow-derived stem cells from a compatible living-related orunrelated donor of a solid organ may be of superior quality and quantitycompared to the recipient's own stem cells.

(e) Bone marrow-derived stem cells alone from a compatible living donorof bone marrow only, and not a solid organ, may be of superior qualityand quantity compared to that of the recipient's own stem cells.

(f) The immediate treatment with allogeneic MSC and/or cells derivedtherefrom by pre-differentiation, provides additional time to harvestand process the patient's own stem cells for subsequent treatments thatmay be needed.

Co-administration of MSC and HSC for a therapeutic dose of stem cellsincludes simultaneous administration of MSC and HSC, administration ofMSC followed by administration of HSC and administration of HSC followedby administration of MSC. For additional therapeutic doses, the timeinterval between the sequential or repeated administration of HSC and/orMSC, respectively, is generally, if utilized, 1-2 days or a few weeks,depending on the responses that are obtained or expected. The stem cellsmay be delivered to the patient as a single population or together asmixed populations given in a single dose. The mixed populations of cellsmay include, but are not limited to any of the stems cells, includingHSC, MSC, pre-differentiated stem cells, hemangioblasts, tubular cells,endothelial cells and combinations thereof. The stem cells may also bedelivered to the patient sequentially. A dose of stem cells may also bedelivered simultaneously or sequentially with a stem cell mobilizationfactor.

In certain embodiments, a therapeutically effective dose of stem cellsis delivered to the patient. An effective dose for treatment will bedetermined by the body weight of the patient receiving treatment, andmay be further modified, for example, based on the severity or phase ofthe kidney or other organ dysfunction, for example the severity of ARF,the phase of ARF in which therapy is initiated, and the simultaneouspresence or absence of MOF. Preferably, about 0.01 to about 5×10⁶ cellsper kilogram of recipient body weight will be administered in atherapeutic dose, more preferably about 0.02 to about 1×10⁶ cells perkilogram of recipient body weight will be administered in a therapeuticdose. The number of cells used will depend on the weight and conditionof the recipient, the number of or frequency of administrations, andother variables known to those of skill in the art. For example, atherapeutic dose may be one or more administrations of the therapy. Asubsequent therapeutic dose may include a therapeutic dose of HSC andMSC, HSC alone, or MSC alone.

The ratio of HSC to MSC for administration for treatment may be greaterthan about 3:1, greater than about 4:1, greater than about 5:1, greaterthan about 6:1, greater than about 7:1, greater than about 8:1, lessthan about 8:1, less than about 7:1, less than about 6:1, less thanabout 5:1, less than about 4:1, less than about 3:1, about 1:1, morepreferably in the range of about 3:1 to about 8:1, most preferably about5:1. Different ratios from those above may prove more effective atcertain stages of ARF, e.g. early vs. late after onset. Different ratiosmay be used for treatment of different or more complex disorders,including MOF. Ratios of may be about 0.1:1 to about 50:1, depending onthe disease being treated.

The therapeutic dose of stem cells will be administered in a suitablesolution for injection. Solutions are those that are biologically andphysiologically compatible with the cells and with the recipient, suchas buffered saline solution or other suitable excipients, known to oneof skill in the art. The stem cells will be delivered at rate known toone of skill in the art.

In another aspect of the present invention, the cellular repairprocesses in ARF or MOF may be significantly accelerated when the cellsadministered to the patient are pre-differentiated in vitro from HSCand/or MSC, as described above. Administration of vascular endothelialcells exerts renoprotective effects in ischemic ARF. HSC and MSC candifferentiate into both renal tubular and vascular endothelial cells,described in Example 13, and into glomerular cells. The cellular repairprocesses may be further accelerated when administered cells arepre-differentiated in vitro (from HSC and MSC) to precursor cells,mature endothelial, renal tubular or cells of other organs. Usingpre-differentiated cells, an injury of kidney or other organs may beorgan- and cell-specifically treated. Organ injury, includingmicrovascular and parenchymal injury, is associated with a significantlevel of HSC mobilization. In both multi-organ failure and ARF, the lowlevel mobilization of HSC may be inadequate to effectively aid in theprotection and repair of severely injured organs. Therefore, replacementof vascular endothelial cells, derived from HSC and/or MSC, combinedwith organ-specific pre-differentiated renal or other parenchymal cellsmay be highly effective in improving organ function and patient/animalsurvival in MOF. Cells for administration for treatment of MOF will bechosen based on the organ exhibiting the most life threateningdysfunction.

In another aspect of the present invention, autologous or allogeneichemangioblasts, a subgroup of HSC and a common stem cell for both bloodand blood vessel cells may be used. Hemangioblasts may be selected byFACS and used for the treatment of MOF, acute renal failure of nativekidneys, ARF of native kidneys in multi-organ failure, and ARF intransplanted kidneys and failure of transplanted organs. Ischemic injuryof various organs results in the spontaneous appearance ofhemangioblasts through their mobilization from the bone marrow into theperipheral circulation. Human hemangioblasts express a characteristiccell surface antigen (CD 133 or AC 133), often in conjunction with CD34, a common stem cell marker, allowing their enrichment with FACSsorting. In mice and rats, vascular endothelial cell precursors orhemangioblasts express the KDR receptor for Vascular Endothelial GrowthFactor (VEGF), also facilitating enrichment by FACS sorting. Upondifferentiation into endothelial or hematopoietic cells, CD 133 and KDRexpression disappears. Hemangioblasts are capable of supporting bothvasculogenesis/angiogenesis and hematopoiesis. These characteristics maybe particularly desirable when there is severe vascular injury of thekidneys and other organs, and poor wound healing.

In another embodiment of the present invention, the pluripotent stemcells may be derived from non-hematopoietic sources such as umbilicalcord blood or other tissue sources, such as the liver, muscle, or fat,or any tissue suitable as a source of pluripotent stem cells. Thenon-hematopoietic stem cells may be enriched in vitro and thenadministered to the patient as described above for the hematopoietic ormesenchymal stem cells. Non-hematopoietic stem cells may be used totreat patients having ARF, TA-ARF, or CRF.

In another embodiment of the present invention, the patient's own stemcells may be used to treat kidney dysfunction by mobilizing endogenousstem cells from the bone marrow. The stem cells may be mobilized withgranulocyte-colony stimulating factor (G-CSF), and/or stem cell factor(SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF),macrophage colony-stimulating factor (M-CSF), interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),interleukin-11 (IL-11), interleukin-12 (IL-12), Flt3-L, TPO and EPO, orany stein cell mobilization factor known to one skilled in the art. Atherapeutic dose of a stem cell mobilization factor is a dose thatincreases the number of circulating stem cells by about 100 fold orgreater, as assessed by the number of CD 34 positive cells in thecirculation. Treatment of patients with stem cell mobilization factorsresults in the mobilization and transfer of hematopoietic andnon-hematopoietic stem cells into the circulating blood. Thus, the bloodthat perfuses the kidneys or other injured organs is enriched with stemcells that are immediately available to protect kidney tubular andvascular endothelial cell functions and survival and that cansubsequently physically replace cells that are damaged or destroyed dueto ischemic or toxic insults to the kidney and other organs. Sinceinjured organs, unlike intact organs, generate homing signals viaactivation of their cognate receptors that attract stem cells, this formof therapy is optimally focused on the protection and repair of adamaged kidney or other organ. A preferred stem cell mobilization factordoes not simultaneously increase peripheral neutrophil numbers, causinggranulocytosis, when the stem cells are mobilized for treatment of apatient having kidney or other organ injury. Studies have demonstratedthat the marked granulocytosis that is associated with stem cellmobilization protocols that use G-CSF plus SCE or cyclophosphamidecauses significant worsening of morbidity and mortality in animals withARF and must thus be avoided. However, G-CSF and SCF or cyclophosphamidemay be used to mobilize and procure stem cells in a patient with normalrenal function in preparation for undergoing a major operation ortherapy that puts the patient at high risk for ARF or MOF andadministered if necessary if ARF or MOF develops. Increased neutrophilsin normal patients do not have the same adverse effects as in patientswith organ injury.

in Pre-Clinical Studies, several methods are used to track administeredHSC, MSC, and EC in the kidney and other organs such as the liver,spleen, lungs, bone marrow, heart or brain. Cell tracking systems areused in which HSC, MSC and EC are labeled with vital dyes prior toadministration. These vital dyes, i.e., dyes that have no harmful effecton living cells, allow the precise location of administered HSC, MSC andEC in the kidney or any organ, using techniques commonly known in theart. Another system that is utilized to track administered cells inexperimental models uses HSC, MSC and EC from syngeneic animals that aretransgenic for human Placental Alkaline Phosphatase (hPAP) or enhancedGreen Fluorescent Protein (eGFP). The administered HSC, MSC and EC fromtransgenic donor animals can be readily identified in wild-typerecipients of the same animal strain, using techniques commonly known inthe art for identifying cells expressing hPAP or eGFP. HSC, MSC and ECderived from a male animal or male human donor, and when these areadministered to a female animal or female human recipient may also betracked. The presence of the male “Y” chromosome in the donor cells thatare engrafted in the recipient's target organs or found in thecirculation can be specifically identified in solid organs byFluorescent In Situ Hybridization (FISH assay), and in general by RT-PCRand immunocytochemistry.

Post-infusion differentiation of HSC, MSC and EC into kidney-specific orother defined cells of injured organs may also be confirmed inpre-clinical studies using the tracking systems. For example, in thekidney, demonstration that the infused HSC, MSC and EC havedifferentiated into the renal cell type that needs to be reconstitutedis accomplished by assaying for the de novo expression of cell markersthat are specific for distinct kidney cell types, e.g., proximaltubular, microvascular endothelial and glomerular cells, respectively.This double labeling technique, i.e., cell tracking and proof ofdifferentiation into organ-specific target cells, provides conclusiveevidence as to the origin (HSC, MSC and EC) and kidney-specificphenotype (proximal tubular, vascular endothelial or glomerular cells)that these cells have differentiated into, respectively. Suitabledifferentiation markers for proximal tubular phenotype are megalin andaquaporin-1, and others. Suitable markers for vascular EC phenotype areCD31 (Pecam-1), von Willebrandt Factor, eNOS, VEGF-Receptor 2,dil-Ac-LDL uptake, and others.

In the bone marrow, as well as in long-term in vitro cultures, MSCsupport growth of HSC and HSC interact with MSC. Both cell types arecapable of differentiation, to a variable degree, into non-hematopoieticcell types, including renal, vascular, neuronal, myocardial, hepatic andothers. Co-administration of these mutually supportive MSC and HSCconceptually reproduces the situation in the bone marrow, potentiallyfacilitating more efficient homing, engraftment and differentiation ofthese cells into those that are dysfunctional or destroyed in ARF, i.e.,delivering humoral factors directed by homing signals to the sites ofinjury that augment cell survival, improve local perfusion, and reduceinflammation, and contributing to the repair of microvascular andtubular injuries.

Microenvironmental changes that are created by vascular and tubular cellinjury in ARF generate homing and differentiation signals for stemcells, and signals that guide and regulate the repair processes, thoughtto be primarily carried out by surviving renal cells. Administration ofMSC and/or HSC, through transient but immediately active mechanisms,protects organ function and augments organ repair. These cells canlocally release growth factors and cytokines such as Hepatocyte GrowthFactor (HGF), Vascular Endothelial Growth Factor, Nitric Oxide, andothers, which are known to exert powerful renoprotective actions. Para-and endocrine intrarenal release of growth factors and cytokines, may beparticularly beneficial in the early phase of ARF treatment with stemcells, since growth factors can enhance cell survival and stimulateproliferation of renal cells in ARF. Subsequent progressivedifferentiation of MSC and HSC into kidney-specific cell types engraftedat sites of renal injury, will directly contribute to or undertake thenecessary cellular repairs.

In addition to providing kidney precursor cells in the treatment of ARF,administered MSC and/or HSC may be utilized for therapeutic genedelivery. MSC, HSC, EC, hemangioblasts or mixes thereof may begenetically engineered or modified by transfection in vitro to augmentthe expression of therapeutically beneficial genes and/or to suppressthe expression of harmful genes prior to administration of cells to apatient. For cell transfections, methods known to one of skill in theart will be used. See e.g. Sambrook et al. Molecular Cloning: ALaboratory Manual (current edition). The transfected genes may includegenes whose products are known to support cellular survival, stimulatecell migration and proliferation, to exert anti-inflammatory andanti-thrombotic actions and to improve intrarenal hemodynamics, andother organ protective effects. The activity of such beneficial genesdelivered in this fashion may be placed under the control ofdrug-sensitive promoters that allow both controlled activation andinactivation of these genes. Genetically engineered cells may be used intreatment of kidney dysfunction, as well as in MOF, organ dysfunction,and wound repair. The term “genetically modified” as used herein refersto stem cells that have been genetically modified with exogenous DNA orRNA. The term “transformed” refers to cells that have acquired malignantcharacteristics. The term “non-transformed” refers to stem cells thathave not been genetically modified with exogenous DNA or RNA.

Defined patient populations are expected to benefit from theadministration of HSC, MSC, EC, hemangioblasts or combinations thereof.For example, patients with treatment-resistant (hemodialysis, parenteralnutrition, antibiotics, ICU care) forms of ARF alone or in the settingof MOF or multi-organ dysfunction, have only a small survival chance andwill therefore be prime candidates for this cell-based treatment.Patients at highest risk for or who are about to develop the most severeform of treatment-resistant ARF would be prepared for HSC, MSC, EC,hemangioblasts or combinations therapy by obtaining their bone marrowaspirate and preparing HSC, MSC, EC, or hemangioblasts as above. Bloodgroup matched, allogeneic MSC or precursor EC or tubular or other cellsderived from MSC by pre-differentiation would be used as the firstintervention in these patients. Time permitting and if no clinicalcontraindications exist, HSC may be obtained with a stem cellmobilization and leukapheresis protocol as detailed above, or by the useof a mobilizing factor such as erythropoietin that only enhance thenumber of peripheral stem cells and EC precursor cells, and notneutrophils that may be harmful to the patient with ARF or MOF. Timepermitting and if needed, autologous MSC may be obtained from thepatients' bone marrow, placed in suitable culture for enrichment andexpansion. These autologous MSC would be administered following theinitial administration of allogeneic MSC and/or precursors, when needed.The prepared autologous stem cells can be cryopreserved and administeredwhen warranted by a poor response to allogeneic MSC or the developmentof life threatening deterioration in the function of kidneys and/orother vitally important organs, i.e., complications that would warrantadditional MSC treatments.

Trauma or surgical patients scheduled to undergo high risk surgery suchas the repair of an aortic aneurysm, may also benefit from prophylacticHSC, MSC, and hemangioblast collection and preparation, from MSC, ofprecursor EC or tubular or other cells, prior to major surgery. In thecase of poor outcome, including infected and non-healing wounds,development of MOF post surgery, the patient's own HSC, MSC, andhemangioblast or MSC-derived target cells that are cryopreserved may bethawed out and administered as detailed above. Patients with severe ARFaffecting a transplanted kidney may either be treated with HSC, MSC, andhemangioblast or MSC-derived precursor cells from the donor of thetransplanted kidney (allogeneic) or with cells from the recipient(autologous). Rejection of both allogeneic HSC or hemangioblasts as wellas that of the transplanted kidney or other organ graft would beprevented by the concomitant administration of immunosuppressive agentssuch as drugs and immune modulating MSC. Blood group matched, allogeneicMSC are an immediate treatment option in patients with TA-ARF and forthe same reasons as described in patients with ARF of their nativekidneys.

In another aspect of the present invention, MSC and/or HSC may beco-administered in defined ratios for the treatment of MOF and the ARFthat always develops in patients with MOF. MSC and/or HSC therapy, inthe setting of MOF, contributes to and augments the defense and repairprocesses of all significantly injured organs, i.e., kidneys, lungs,heart, liver, etc. It has been shown that the administration of bonemarrow-derived stem cells to animals with damage of very differentorgans, e.g., experimental stroke or neurotoxic insult models, spinalcord injury, myocardial infarction, liver injury, and ARF data describedbelow, results in protection and repair of individually targeted organs.Co-administration of MSC and HSC and/or other cells, as described inthis invention, may thus represent an intervention that can effectivelyboost both a patient's capacity to survive the immediate deleteriousconsequences of MOF and to subsequently carry out repair of organs thatare damaged in the context of MOF.

In another aspect of the present invention, MSC, HSC, precursor cellsderived from MSC or the bone marrow or circulation, orpre-differentiated cells specific to the target organ and combinationsthereof may be administered in defined ratios for the treatment of ARFin the transplanted kidney. MSC have been shown to act in animmunomodulatory manner, i.e., they are able to enhance a recipient'stolerance for an allograft (see above). Administration of bonemarrow-derived allogeneic stem cells from the kidney donor results ingeneralized microchimerism in the kidney recipient, also known to leadto enhanced graft tolerance. Co-transplantation of MSC and HSC may haveimmediate renoprotective effects, as in ARF of native kidneys (seeabove), thereby ameliorating or preventing EGD or TA-ARF, as well asdiminishing the late consequences of severe EGD (increased graftrejection rates) by induction of enhanced graft tolerance throughseveral immune-modulatory mechanisms (see above).

Administration of autologous HSC, MSC, MSC-derived precursor cells ororgan-specific target cells and combinations thereof obtained in advanceto the kidney transplant from the kidney recipient, may permitsignificant replacement of donor renovascular endothelial cells withthose of the recipient. Replacement of donor renovascular endothelialcells that are lost in EGD with endothelial cells prophylacticallyobtained from the recipient, or derived by pre-differentiation fromautologous MSC and/or HSC, may thus reduce the immunogenicity of thedonor kidney, since vascular endothelial cells represent the mostimmediate barrier between the recipient's blood elements, includingcells and antibodies that mediate vascular/cellular rejection, and theparenchymal cells of the implanted kidney. Replacement of a significantpercentage of the donor kidney's vascular endothelial cells withendothelial cells derived from the recipient, the recipient's MSC and/orHSC will reduce the number of “foreign” vascular endothelial cellspresent in the transplant, creating a “renovascular microchimerism”.

Stem cells are administered to the patient by infusion intravenously(large central vein such vena cava) or intra-arterially (via femoralartery into supra-renal aorta). Any delivery method for stem cells,commonly known in the art, may be used for delivery of the administeredMSC, HSC, hemangioblasts or precursor cells obtained bypre-differentiation from MSC as defined above.

In certain embodiments, a therapeutically effective dose of stem cellsand/or a therapeutically effective dose of a stem cell mobilizationfactor(s) that does not cause a rise in peripheral neutrophils aredelivered to the patient with ARF, TA-ARF, or MOF. An effective dose fortreatment will be determined by the body weight of the patient receivingtreatment, the patient's response to these treatments, comorbidities andseverity of disease. A therapeutic dose may be one or moreadministrations of the therapy. Delivery of the stem cells may be bymobilization of endogenous stem cells, or by intravenous orintra-arterial infusion.

In another aspect of the present invention, the above delineatedtechnologies may be established in tertiary care centers world wide. Inanalogy to company-owned in-hospital and free-standing hemodialysisunits, multidisciplinary “Stein Cell Nephroplasty Teams” or “CellTherapy Teams” could be owned and operated by international Health CareEquipment and Service companies that would also produce and sell theiror other companies' kits and materials used for the harvesting,purification, culturing, differentiation, cryopreservation, thawing,quality control and administration of stem cells or stem cells that arepre-differentiated in vitro to patients at high risk for ARF ormulti-organ failure (Prevention), and to patients with established ARFor multi-organ failure (Treatment). Physicians (Nephrologists.Intensivists, etc.) who care for this group of patients would orderrespective cell-based services, and these specialized teams wouldprovide the requested treatment.

In a preferred embodiment of the present invention, the stem cells (HSC,MSC, hemangioblasts, precursor cells) utilized for these treatments willbe “harvested” and prepared on site, i.e., in the hospital by aspecialized team or in free-standing “Stem Cell Donation Centers” fromthe following donors: 1) a patient will donate his/her own bone marrowfor treatment of his/her own ARF or organ failure, i.e., autologous stemcells; 2) a blood group and tissue-type identical allogeneic donor; 3) ablood group compatible but not tissue-type identical allogeneic donor assource of allogeneic MSC and precursor cells derived from these MSC.Stem cells from these donor groups are administered when ARF develops inthe native or transplanted kidney or when organ failure develops inanother transplanted organ (heart, liver, lungs, pancreas, islet cells,and others). In this setting, harvesting of solid organs from acadaveric donor (kidneys, liver, heart etc.) would be complemented bythe simultaneous harvesting of the cadaveric or living related donor'sbone marrow-derived stem cells, by that very same specialized team (asabove). Since the solid organs to be transplanted are always screenedfor compatibility with prospective recipients, the simultaneouslyharvested stem cells would thus be automatically identified as beingcompatible with the recipient of any of the solid organs. Thus, keepingthese stem cells available by cryopreservation, makes them readilyavailable for developing treatment needs following transplantation ofthe solid organs into multiple recipients (kidneys, heart, liver, etc.).The immunosuppressive drugs needed to prevent rejection of atransplanted organ (kidney, heart, liver, lungs, pancreas, etc.) wouldsimultaneously serve to prevent the rejection of administered,non-tissue type identical stein cells. This effect would be furtherenhanced by the immune modulating actions of MSC, if co-administered. Asused herein, the terms “harvesting and administering” may include thefollowing: harvesting, processing, enriching, characterizing,cyropreserving, thawing, performing quality control, and administering.

In another embodiment of the present invention, the horning signals andmechanisms that direct the delivery of stem cells to the sites of injuryin the native or transplanted kidney with ARF, with CRF or to otherdamaged organs in MOF or to poorly healing wounds may be utilized.Injured organs upregulate expression of SDF-1 alpha and SDF-1 beta,chemokines that attract stem cells and precursor cells expressingCXCR-4, the receptor for SDF-1. At the injury site, locally producedSDF-1 results in the generation of a concentration gradient.Consequently, the SDF-1 concentration is highest at the site of tissueinjury and determines thus the direction and intensity of a stem cellshoming response. The stern cells and precursor cells of the presentinvention may be genetically modified to optimize the expression ofCXCR-4 on their cell surface to thereby increase the homing response ofthe cells to the injury site. Similarly, additional receptors present onstem cells known to one of skill in the art, i.e., receptors thatadditionally mediate the homing of stem cells to injured tissues may beover-expressed by transfection in the stem cells, precursor cells, ortarget cells of the present invention. Transfection methods known to oneof skill in the art may be used to genetically modify the cells tooptimally home to the site of injury.

In another embodiment of the present invention, the bone marrow stemcell mobilization signals and mechanisms that emanate from a kidney withARF or from other injured organs can be augmented for therapeuticindications by the administration of stem cell mobilization factorsknown to those skilled in the field. In this fashion the spontaneousstem cell mobilization response that appears inadequate to protectseverely injured kidneys or other organs can be augmented by suitablefactors such as erythropoietin.

EXAMPLES Example 1 Determine the Relative Renoprotective Potency of HSC,MSC, Precursor Vascular Endothelial or Tubular Cells Derived from MSC byPre-Differentiation, Hemangioblasts, of Fully Differentiated VascularEndothelial Cells, and of Fibroblasts Administered to Rats with ARF

In experiments, adult Sprague-Dawley or Fisher 344 rats (male or female)were studied. Ischemia/reperfusion-type of ARF (“ischemic ARF”) isinduced in anesthetized rats by timed clamping of both renal pedicles,thereby interrupting the blood supply to the kidneys causing an“ischemic” insult that results in acute loss of kidney function, i.e.,ARF. A model of severe ARF in rats used 45 minutes of bilateral renalischemia, resulting in a rise in serum creatinine to 3.5-5.0 mg/dL, aglomerular filtration rate of <15% of normal, and a mortality of 50% at72 hrs post reflow. Histological examination of the kidneys from thissevere ARF model shows wide spread tubular necrosis, apoptosis andsevere vascular congestion with accumulation of inflammatory cells inthe corticomedullary junction. A moderate ARF model in rats used 35minutes of bilateral renal ischemia, resulting in a rise in serumcreatinine level to 1.5-2.5 mg/dL at 24 hours post reflow, and amortality of <10% at 72 hours post reflow. Histological examination ofthe kidneys from this moderate ARF model demonstrates more limitedtubular necrosis, apoptosis and modest vascular congestion with lowerlevel accumulation of inflammatory cells in the corticomedullaryjunction. These models of ARF very closely resemble the most common andmost serious forms of ARF encountered in patients with shock, sepsis,trauma, after major cardiac or vascular surgery, etc.

The relative renoprotective potency of various SC and cell treatmentprotocols was tested by infusing intravenously (jugular, femoral or tailvein) or intra-arterially (into suprarenal aorta via carotid or femoralartery) syngeneic HSC alone, MSC alone, precursor cells of endothelialor tubular phenotype obtained by pre-differentiation of MSC,hemangioblasts, obtained from HSC by FACS sorting, mature vascular ECand, as cell control, fibroblasts either immediately or 24 hrs afterinduction of severe or modest ARF, respectively. The total number ofeach cell type administered in all studies was about 10⁵ to 10⁶cells/animal, Control animals were sham operated and were injectedeither with vehicle or fibroblasts alone.

Renal function in all animal groups was monitored, as in patients, bydetermination of blood creatinine and BUN levels, serum electrolytes,measurement of renal blood flow (ultrasonic method), creatinine andinulin clearances and urine output. Overall animal outcome was assessedby determination of weight loss, hemodynamics (blood pressure), andsurvival. After sacrifice of control and cell-treated animals with ARFand shams, kidneys were examined for the degree of histological injury(cell apoptosis, necrosis, vascular congestion and injury, inflammatorycell infiltrates) and repair (mitogenesis, redifferentiation of cells,decongestion, etc.), intrarenal localization of the administered HSC/MSC(as discussed above, the administered HSC/MSC are tagged for trackingpurposes), and their integration and differentiation into renal cells.Selected animals in the various groups were followed for up to 28 daysafter start of study.

The following observations from the experiments using the rat model ofARF were made: (1) all types and sources of administered EC (precursorsfrom MSC, mature EC) and hemangioblasts significantly protect renalfunction and improve outcome in rats with ischemic AU, both when givenimmediately or 24 hrs post reflow. Protection in all groups wassignificant both in animals with severe and modest ARF, the cell typethat appears most protective when administered immediately after reflowof the ischemic kidneys appears the EC phenotype, however, thesubsequent renoprotective effects obtained with the administration ofall individual cell types were comparable. In addition, mortality insevere ARF is reduced from ˜40% to <5%; (2) MSC administered aloneresult in delayed but significantly accelerated recovery of renalfunction; (3) HSC infused alone show similar or slightly lessimprovement in functional recovery compared to that obtained with MSC orEC; (4) fibroblast infusion had no effect on renal function in rats withARF or shams. Obtained functional protection and return of functionshows good correlation with histological injury scores as defined above.Administered tagged cells are readily detected in the microvasculatureof ARF kidney but not in kidneys from sham animals or in their urine. Byday 7 following infusion, the tagged cells appeared phenotypicallyunchanged. Additionally, there were no adverse effects that resultedfrom the administration of any cell type in sham or ARE animals, likelybecause these were autologous (from the animal or potentially thepatient who is treated for ARF) or “syngeneic” cells (from the animal'slitter mate or a HSC/MSC donor who is immune-compatible with the patientwho is treated for ARF). Importantly, additional in vivo studies withallogeneic MSC demonstrated that these were well tolerated and exhibitedrenoprotective effects in rats with ARF that were identical to thoseobtained with syngeneic MSC.

The results from the experiments in the rat models will be applicable tothe treatment of patients. In clinical practice, patients who qualifyfor this form of treatment, i.e., those with the severest form of ARF,one that carries a mortality of up to 100%, particularly when ARFdevelops in the setting of multi-organ failure, may serve as their own,autologous HSC, MSC, EC, or hemangioblast donors. Accordingly, bonemarrow is aspirated under local anesthesia and under sterile conditions.HSC are isolated and enriched from the bone marrow aspirate using FACSand are subsequently cryopreserved until use.

Hemangioblasts are obtained form HSC by FACS sorting for CD 133. Highlypure MSC are generated in sterile culture of bone marrow aspirates, arecryopreserved until needed, or are pre-differentiated into EC andtubular cell precursors, and cryopreserved until needed. This autologousapproach requires, however, that the patient who is in need of this formof treatment is able to survive for the number of days that are neededto harvest, enrich, culture expand, differentiate, etc. his/her owncells. Because of this time delay, the vast majority of patients withsevere ARF or MOF will not be able to be treated with their own cells,unless these have been procured prior to the development of ARE or MOF.If not available, allogeneic MSC that are blood group compatible, intheir undifferentiated state or after pre-differentiation into EC ortubular phenotype are administered as fist line treatment, exploitingtheir robust immunomodulating capacity. Subsequent SC or cell treatmentsmay be repeated allogeneic MSC doses or autologous SC that have beenprocured from the patient in the mean time.

Additional studies will be conducted to determine whether theadministration of a single cell type (see above), repeatedadministrations of a single cell type or cell combinations are morerenoprotective. A particular focus in these experiments will be todetermine if co-administered HSC and MSC represent a superior form oftherapy for the specific conditions that are treated with the presentinvention (Example 2 below).

Example 2 Determine the Ratio of MSC and HSC for Co-AdministrationTherapy

Using as a guideline the approximate ratio of HSC and MSC numbers in thenormal bone marrow, protocols in which the ratios or doses ofco-administered HSC/MSC given to rats with ARF, models and animalstrains as in Example 1, were varied.

The relative renoprotective potency of various SC treatment protocolswas tested by infusing intravenously (jugular, femoral or tail vein) orintra-arterially (into suprarenal aorta via carotid or femoral artery)HSC alone, MSC alone or HSC in combination with MSC at a HSC/MSC ratioof 1:1, 3:1, 5:1 or 8:1 to rats immediately after induction of severe ormodest ARF as well as infusion of HSC alone, MSC alone or HSC/MSC inratios of 1:1, 3:1, 5:1 or 8:1 24 hrs after induction of severe ormodest ARF in rats (see above). The total number of cells administeredin all studies was about 10⁵ to 10⁶ cells/animal.

Renal function, histological studies and outcomes in the experimentalmodels were monitored as detailed in Example 1 above.

The following observations from the experiments using the rat model ofARF were made. Outcome is greatly improved when HSC/MSC are administeredin combination at an average HSC/MSC ratio of 5:1. Animal mortality wasabolished with the combination treatment. In comparison, HSC or MSCgiven individually provide a modest to good renoprotective effect. Stemcell homing, subsequent engraftment and gradual differentiation andintegration into the ARF kidney occurred with much greater efficiencywhen HSC and MSC were co-administered, likely explaining the excellentorgan repair and functional recovery that is obtained. Additionally,there were no adverse effects that resulted from the administration ofHSC, MSC or both together.

Additional studies will be conducted to optimize co-administrationprotocols, including using different ratios of stem cells, includingallogeneic stem cells, and to further identify and augment intrarenalhoming and differentiation signals.

The results from the experiments in the rat model will be applicable tothe treatment of patients. In clinical practice, patients who qualifyfor this form of treatment, i.e., those with the severest form of ARF,one that carries a mortality of up to 100%, particularly when ARFdevelops in the setting of multi-organ failure, will serve as their own,time permitting, autologous HSC/MSC donors. Bone marrow is aspiratedunder local anesthesia and under sterile conditions. HSC are isolatedand enriched from the bone marrow aspirate using FACS and aresubsequently cryopreserved until use. Highly pure MSC are generated insterile culture of hone marrow aspirates. Appropriate numbers of HSC andMSC are combined at a defined ratio, e.g., 5:1, suspended in sterilesaline or McCoy' solution, and administered into a large central vein.The latter access is always established in this group of patients.Unless contraindicated, a suprarenal aortic route of administration mayprove superior, and can be routinely accomplished by cannulating afemoral artery and advancing the tip of the infusion catheter to anintra-aortic location well above the renal arteries. This route ofadministration allows the most direct and SC dose-sparing delivery ofHSC/MSC into both renal arteries and thus into both kidneys. Studies inwhich the therapeutic results that are obtained with the intravenousinfusion route (superior vena cava) are compared with those obtainedusing the intra-aortic route will establish which approach is superior.It is also important to note that, if needed, treatments with autologousHSC/MSC can be repeated. And, HSC and MSC from the donor of a kidneywhose tissue type is close enough to that of the recipient and thuspermits a successful allogeneic transplant, thereby requiring no or onlymodest immunosuppresive therapy, may also be administered at the time ofor following the kidney transplant, for the treatment of EGD,respectively. Importantly and as detailed in Example 1, allogeneic MSCor their derivatives may be co-administered with autologous HSC, sincethe latter can be procured more quickly.

Example 3 Determine the Relative Potency for Wound Healing of HSC, MSC,Precursor Vascular Endothelial or Tubular Cells Derived from MSC byPre-Differentiation, Hemangioblasts, of Fully Differentiated VascularEndothelial Cells, and Define the Optimal Ratio of MSC and HSC forCo-Administration for Wound Healing

The administration of individual cell types, as above, or MSC and HSCmixes to rats with ARF resulted in improved outcome (see above). Also,the abdominal, well-healed incision initially created for the inductionof ARF (clamping of both renal arteries), contained large numbers (˜40%)of tagged MSC and HSC-derived vascular and other cells, indicating thatMSCs and HSCs can powerfully support the process of wound healing thatincludes angiogenesis. Further studies in animals with experimentalabdominal wound infections alone or in the setting of LPS-induced shockwith MOF, or in rats with combined ischemic ARF and cecalperforation-induced peritonitis/sepsis will examine whether celltherapy, as defined above, improves wound healing and related outcomes(see Example 4).

Example 4 Determine Stem Cell Therapy Protocols for Multi-Organ Failure

Stem cell therapies will be investigated that may effectively boost thebody's ability to cope with the many deleterious consequences ofmulti-organ failure and to carry out repair and functional recovery ofmultiple organs rather than that of a single one such as the kidney withARF. The multi-organ failure models that will be used is the endotoxinmodel in mice, in which endotoxin from gram negative bacteria (LPS) isinjected, resulting in many manifestations of clinical multi-organfailure, including ARF. Another model of MOF in rats or mice combinesischemic ARF and cecal perforation-induced peritonitis/sepsis, shown tomost optimally reproduce the manifestations of clinical MOE Besidesimprovement in organ function, successful MSC and HSC therapy isexpected to reduce the 100% mortality seen in experimental multi-organfailure, and to significantly enhance wound repair, when applicable (seeExample 3 above).

Example 5 Determine MSC, HSC, and EC Therapy for GeneralizedMicrochimerism

Interventions to establish generalized microchimerism in order to induceincreased immune tolerance of the transplanted kidney or other organs,i.e., reduced rejection rates, will be examined using suitable rat andmouse kidney transplant models, and employing autologous and allogeneicdonor and recipient combinations. The HSC and/or MSC will beadministered alone or in various ratios. HSC/MSC pre-differentiated invitro or hemangioblasts will also be administered in separateexperiments. The degree of microchimerism is determined byidentification of tagged donor cells in the circulation, bone marrow andkidney, when applicable. The degree of graft acceptance or tolerance istested in animals with allogeneic transplants by tapering ordiscontinuing antirejection medications. Animals with microchimerism areexpected to exhibit lower rejection rates than those without. MSC and ECmay also be used to establish a state of “microchimerism”. The uniqueimmunomodulating effects of allogeneic MSC and EC precursors that arederived from MSC, as above, may prove particularly beneficial for themanagement of TA-ARF or EGD and for the boosting of graft survival fortransplanted organ in general.

Example 6 Determine MSC, HSC, and EC Therapy for “RenovascularMicrochimerism”

Interventions to establish “renovascular microchimerism” in order toinduce increased immune tolerance of the transplanted kidney, i.e.,reduced rejection rates, will be examined using suitable rat and mousekidney transplant models, and employing autologous and allogeneic donorand recipient combinations. The HSC and MSC will be co-administered invarious ratios. HSC/MSC pre-differentiated in vitro, hemangioblasts, orEC or combination thereof will also be administered. The degree ofmicrochimerism is determined by identification of tagged donor cells inthe circulation, bone marrow and kidney vasculature. The postulateddegree of enhanced graft tolerance as a function of “renovascularmicrochimerism” is assessed as in Example 5 above. The uniqueimmunomodulating effects of allogeneic MSC and EC precursors that arederived from MSC, as above, may prove particularly beneficial for themanagement of TA-ARF or EGD and for the boosting of graft survival ortolerance.

Example 7 Determine Therapeutic Effectiveness of Hemangioblasts in ARFand MOF

Following the experimental design protocols detailed above,hemangioblasts isolated from bone marrow harvested, FACS enriched HSCwill be administered to prevent or treat ARF (native kidneys,transplanted kidney) and multi-organ failure. The very high potential ofthese cells to differentiate into vascular endothelial cells may proveto be particularly advantageous when renovascular or generalizedvascular injury predominates in a particular phase of ARF of multi-organfailure. Results obtained with hemangioblasts will be compared to thoseobtained with protocols detailed in the preceding Examples.

Example 8 Examine the Effect of Hematopoietic Stem Cell Mobilization onthe Outcome of Ischemia/Reperfusion-Induced ARF in Rats and Mice

Ischemic ARF will be induced in anesthetized, adult rats and FVB mice bytimed clamping of both renal pedicles and in rats as above. Renalfunction, histological changes, overall outcome will be monitored asabove. Since stem cell mobilization with cytoxan, followed by G-CSFmaximally increases both HSC and neutrophils in the circulation, andsince this protocol causes a marked increase in the mortality of animalswith ARF, such an approach must be avoided clinically. However, HSCmobilization with erythropoietin and other factors is not associatedwith a significant rise in peripheral neutrophil numbers, which suggeststhat such a form of stem cell mobilization may be renoprotective in ARF,TA-ARF and MOF.

Example 9 Characterization of Homing Signals and Mechanisms for Stem andMSC-derived Cells in the Kidney with ARF

The kidneys and HSC, MSC, EC (precursors from MSC, mature EC), andhernangioblasts from the animals studied in Examples 1 and 2 will befurther examined for SDF-1 and CXCR4 expression using in situhybridization, real time PCR, and immuno-histo- and cyto-chemistry. Theimportance of the chemokine SDF-1 alpha, its beta splice variant, andits receptor CXCR4 in mediating chemokinesis of HSC/MSC and other cellswill be investigated in vitro using transwell migration assays and inproof of principle experiments with neutralizing anti-SDF-1 oranti-CXCR4 antibodies. The effect of administered neutralizinganti-CXCR4 antibodies on the homing efficiency of tagged HSC and MSC inthe ARF kidney will be assessed.

Determinations will be made to corroborate that injured tubular orendothelial cells in ARE express SDF-1 (alpha or beta), and whethermobilized stem cells express CXCR4. This determination will provide fora system for mediation of homing of CXCR4-expressing stem cells towardsthe sites of nephron and vascular injury. Homing efficiency of theHSC/MSC/EC and hemangioblasts will be optimized to improve therenoprotective and organ protective stem cell therapies.

Example 10 Determine the Effect of HSC or MSC Therapy on the Outcome ofARF in Mice

In order to determine whether HSC or MSC home into the kidney in ARE,and whether they transdifferentiate, integrate and act renoprotectively,genetically marked, phenotyped cells will be exogenously administeredand traced in the kidney of mice with ARF. HSC or MSC will be obtainedfrom the femurs of eGFP transgenic FVB mice that express enhanced greenfluorescent protein (eGFP+HSC). The eGFP+HSC will, if necessary, befurther enriched by FACS sorting (c-kit, sca-1, lin negative), eGFP+MSCare clonally expanded, and administered intravenously to wild type micewith ARF as described in Examples 1 and 2 above. Appropriate controlswill be included. At defined time points following induction of ARF,kidneys from experimental and control mice will be examined in order toassess where eGFP cells are located and whether they havetransdifferentiated into renal tubular or endothelial cells,respectively. Renal function and histology is examined as above and fordirect tissue evidence of transdifferentiation and integration of eGFP+cells into tubular or vascular endothelial sites at which ARF causedcell injury and loss. The paracrine potential of HSC and MSC to produce,deliver and release renoprotective growth factors and cytokines in situ(e.g. HGF, EGF, IGF-I, VEGF, NOS, and others) will be tested in in vitrostudies using ELISA and other suitable assays and real time-PCR. In vivostudies with neutralizing antibodies to growth factors and cytokines ortheir respective receptors or inhibitors of NOS will be used to test theimportance of these factors as mediators of renoprotection and repair.

Example 11 Determine the Effect of MSC Therapy on Outcome of ARF in Mice

MSCs originate, like the kidneys, from the mesoderm and have been shownto transdifferentiate into numerous cell types. MSC from eGFP transgenicFVB mice will be utilized. The eGFP+MSC will be isolated from harvestedbone marrow based on their characteristic and selective attachment tothe culture dish. A functional MSC culture system will be establishedthat provides for well maintained eGFP expressing MSCs at laterpassages. Cultured eGFP+MSC will be administered to wild type mice withARF as described in above Examples and outcome and tissue analyses willbe performed as above. The results will be analyzed to determine the MSCrenoprotective effects as compared to the HSC renoprotective effects.The results may suggest that co-administration of HSC and MSC may bemost beneficial, since these cells depend on each other for effectivehematopoiesis. The paracrine potential of MSC to produce, deliver andrelease renoprotective growth factors in situ including HGF, EGF, IGF-I,etc. will be tested in in vitro and in viva studies as described inExample 10.

Example 12 Assess the Effect of MSC Therapy on the Function ofRenovascular Endothelial Cells in ARF

The kidney is a highly perfused organ, receiving 20% of the cardiacoutput, and the complexity of the intrarenal circulation facilitates theprocesses of filtration and tubular transport. It is now recognized thatvascular endothelial cell dysfunction and death are importantdeterminants of loss of renal function in ARF. The bone marrow containsendothelial precursor cells (CEP, circulating endothelial precursors),that can be mobilized into the peripheral circulation, from where theycan contribute to wound healing or participate in tumor angiogenesis.Both bone marrow-derived stem cells types, HSC and MSC, are able totransdifferentiate into endothelial cells. The effect of MSC will betested, after transdifferentiation into endothelial cells, orc-kit+/VEGFR2+hematopoietic cells, from eGFP transgenic mice, on thecourse of ARF. The cell type that will be assessed in these experimentsis the postglomerular vascular endothelial cell that is injured orkilled in ARF.

MSC and c-kit+/VEGFR2+ hematopoietic cells from eGFP transgenic mice(CEP) will be subjected to various transdifferentiation protocols invitro with the goal of obtaining endothelial cells, phenotypicallyconfirmed by appropriate endothelial cell markers. Cells will then beadministered to mice with ARE as in the preceding protocols (Examples 1and 2) and their impact on the course of ARF will be monitored as above.Kidney tissues will be examined for location of administered stem cellsand vascular integration.

Example 13 Examine the In vitro Transdifferentiation of MSC into RenalTubular and Vascular Endothelial Cells

Spontaneous transdifferentiation of MSC generally does not occur.Treatment of MSC cultures with specific factors results in theirtransdifferentiation into adipocytes, osteocytes, chondrocytes and othercell types. Differentiation factors will be identified that result intransdifferentiation of MSC into tubular cells. The kidney is ofmesodermal origin and during embryonal nephrogenesis ureteric bud cellsinduce a mesenchymal-epithelial transdifferentiation in the metanephricmesenchyme. This process is influenced by several growth factors (HGF,BCE, LIF, TGF alpha, FGF2) that exhibit redundancy and is critical tooverall nephrogenesis, since failure of induction of the metanephricmesenchyme results in its apoptosis, and since the mesenchyme, on theother hand, induces the ureteric bud to undergo branching morphogenesiswhich results in collecting duct formation. MSCs will be examined todetermine their ability to transdifferentiate into tubular epithelialcells.

Cell culture systems optimized for MSC, including plating of cells oncollagen (I or IV) and/or fibronectin, exposure to differentiationfactors such as VEGF and others, co-culture systems with target cells,conditioned media from target cells, are used in these studies. Thecapacity of various culture conditions, differentiation and growthfactors to induce the transdifferentiation of MSC into renal vascularendothelial cells and tubular progenitor cells will be examined. EC maybe generated in vitro from HSC or MSC by differentiation. For example,MSC may be grown and differentiated in culture, followed by injury, forexample by scraping or ATP depletion. Additional MSC added to theinjured target cells with conditioned media and the MSC then become ECwhich may be used for administration. Tubular cells may be generated bythe same injury model.

Pax-2 will be used as an initial marker of tubular cell induction, sinceit is a kidney specific transcription factor that is expressed in theembryonic kidney, and that, importantly, is re-expressed in proximaltubular cells that are injured in ARF. Megalin or aquaporin-1 aremarkers of proximal tubular cells. PECAM-1 (CD34), von WillebrandtFactor, dil-ac-LDL uptake, eNOS, VEGF-Receptor 2, and others aresuitable markers of EC phenotype.

The in vitro systems for induction of MSC into tubular or endothelialcells will then be used for further analysis of molecular mediatorsignals and their utility for pre-differentiation of MSC into these celltypes that may be subsequently tested in ARF treatment protocols, asdetailed above.

For in vitro differentiation of MSC into EC, MSC may be plated ontoMatrigel® using techniques known to one of skill in the art (availablefrom BD Biosciences, Franklin Lakes, N.J.).

Example 14 Analyze In vivo Transdifferentiation and Integration ofIntrarenally Injected MSC (Subcapsular, Cortical Interstitium) and HSCin Intact and ARF Kidneys in Mice

HSC and MSC from eGFP transgenic mice will be injected (subcapsular orin mid cortex) into normal and ARF kidneys of wild type mice. Theirpotential transdifferentiation and integration into tubular and vascularstructures will be analyzed histologically, using appropriatedifferentiation markers as above. The data from these in vivo studieswill determine whether MSC and/or HSC are able to transdifferentiate invivo into specific renal cell types and the location of the injectedcells. The influence of preexisting injury due to ARF on these processeswill be assessed, and the influence of spontaneously or experimentallyincreased SDF-1 levels at the sites of injury on SC horning isdetermined.

Example 15 Analyze the Effects of HSC, MSC, Precursor Cells (EC, TubularCells), Hemangioblasts and Combinations Thereof on Kidney Function inAnimals with Underlying Chronic Renal Failure or Chronic Kidney Diseaseper se or Superimposed ARF

The effects of HSC and/or MSC and other defined cell treatments, asabove, on the course of CRF (CKD) is examined in a progressive rat modelwith CRF, induced by ⅚^(th) nephrectomy or unilateral nephrectomy andcontralateral, selective renal artery branch ligation. In addition,these cell therapies are also tested in a transgenic mouse model of typeII diabetes mellitus (db/db strain) that develops progressive diabeticnephropathy and CRF is examined. Outcomes over time (renal function,urinary protein excretion, blood pressure, survival, and kidneyhistology) are examined as above in experimental and control animals.

The effects of SC treatment protocols tested in ARF or MOF models(models without underlying renal disease) (see Examples above) on theoutcome of rats and mice with underlying CRF, induced surgically in ratsor in transgenic db/db mice, will be analyzed in order to see whetherstem cell therapy is effective in these high risk for ARF models, aclinically highly relevant issue.

Example 16 Analyze the Effect of Genetically Altered MSC in Animals withARF, TA-ARF, MOF and CRF.

It will be tested whether in vitro induced changes (by transfection) inthe expression of renoprotective growth factors, cytokines, hemodynamicmediators, anti-inflammatory, anti-thrombotic or harmful cytokines ortheir receptors in MSC prior to their administration to animals with AREcan be used to boost the renoprotective potency of these cells. Genes ofthese humorally acting factors may be placed under the control of drugs,allowing for regulated expression or suppression of such cytokines.Outcomes will be tested as in the above Examples.

Example 17 Analyze the Effect HSC on Kidney Allograft Function

The effect of HSC, MSC and/or stem cell mobilization treatments onshort- and long-term kidney allograft function will be analyzed. Kidneytransplantation will be performed using a two-step rat model. The donorwill be a Fisher 344, male rat, transgenic for human placental alkalinephosphatase. The recipient will be a compatible Fisher 344 female wildtype rat or an incompatible Lewis female wild type rat. Function andoutcome studies will be performed as described above using the kidneyallograft rat model.

Although the invention herein has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, modifications, substitutions, and deletionsnot specifically described may be made without departing from the spiritand scope of the invention as defined in the appended claims. It istherefore intended that the foregoing detailed description be regardedas illustrative rather than limiting, and that it be understood that itis the following claims, including all equivalents, that are intended todefine the spirit and scope of this invention.

1. A method of treating multi-organ failure, kidney dysfunction, orwound healing, said method comprising delivering a therapeutic amount ofstem cells to a patient in need thereof.
 2. (canceled)
 3. The method ofclaim 1 wherein said stem cells comprise mesenchymal stem cells. 4-5.(canceled)
 6. The method of claim 1 wherein said stem cells comprisenon-transformed stem cells.
 7. The method of claim 1 wherein said stemcells comprise genetically modified stem cells, wherein protectivepotency of said cells is augmented by genetic modification prior toadministration in a patient in need thereof.
 8. The method of claim 1wherein said stem cells comprise autologous cells.
 9. The method ofclaim 1 wherein said stem cells comprise allogeneic cells.
 10. Themethod of claim 1 wherein said kidney dysfunction comprises earlydysfunction of kidney transplant or chronic renal failure. 11-17.(canceled)
 18. A method of treating multi-organ failure, kidneydysfunction, wound healing or organ dysfunction comprising delivering atherapeutic amount of a stimulant of stem cell mobilization to a patientin need thereof; wherein the stimulant mobilizes stem cells to theorgans in need thereof.
 19. The method of claim 18 wherein saidstimulant of stem cell mobilization is SDF-1.
 20. The method of claim 18wherein said stem cells comprise mesenchymal stem cells. 21-44.(canceled)
 45. A method of treating multi-organ failure, kidneydysfunction, organ dysfunction, or wound healing, said method comprisingdelivering a therapeutic amount of a mixture hematopoietic stem cellsand mesenchymal stem cells to a patient in need thereof.
 46. The methodof claim 45 wherein said kidney dysfunction comprises acute renalfailure, early dysfunction of kidney transplant, or chronic renalfailure.
 47. The method of claim 45 wherein said hematopoietic stemcells and said mesenchymal stem cells comprise autologous cells.
 48. Themethod of claim 45 wherein said hematopoietic stem cells and saidmesenchymal stem cells comprise allogeneic cells.
 49. The method ofclaim 45 wherein a ratio of said hematopoietic stem cells to saidmesenchymal stem cells is optimized for the treatment of kidneydysfunction or other organ dysfunction.
 50. The method of claim 49wherein said stem cells are delivered to said patient in a ratio ofabout 0.1:1 to about 50:1 hematopoietic stem cells to mesenchymal stemcells. 51-59. (canceled)
 60. The method of claim 3, wherein themesenchymal stem cells are isolated from bone marrow aspirates andadhere to the bottom of a culture dish while substantially all othercell types remain in suspension.
 61. The method of claim 45, wherein themesenchymal stem cells are isolated from bone marrow aspirates andadhere to the bottom of a culture dish while substantially all othercell types remain in suspension.